HUT62035A - Process for producing strains of nitrogen-fixing bacteria mutant in nitrogen control of symbiotic nodule-forming genes - Google Patents

Abstract

For the improvement of the expression of nodulation genes in symbiotic nitrogen fixing bacteria at high ammonia concentrations the gene regulating the expression of nodulation genes of the microorganisms is inactivated, preferably by chemical mutagenesis, by inserting a foreign DNA sequence into the gene (insertion, transposition), by removing the gene or a fragment thereof (deletion), or by transferring the mutated gene into other bacterial strains or species (with conjugative or mobilizable plasmids, transducing phages, or after molecular cloning, with any plasmid or phage vector) and replacing the wild type gene by the homologous region of the mutant gene.

Description

The present invention relates to a process for the production of nitric-binding bacterial strains which are mutant in the regulation of symbiotic tuberase genes, which are effective in modulating the presence of bound KxLtpoeéKi (ammonia).

The specific interaction between Rhizobium mellloti bacterium and its host plant, alfalfa, results in the formation of symbiotic nitrogen-binding nodules at the root of the plant. Modified bacteria in the tubers, bacteroids, reduce the molecular nitrogen of the air to anonymis and thus provide the host plant with nitrogen · In exchange, the plant provides the bacterial oocytes with energy and sheds.

Different nitrogen-binding bacterial species establish a symbiotic relationship with different, agriculturally important butterfly-host plants. For example, Rhizobium mellloti with alfalfa (Medicago), R. trifolli with testis (Triflium), R.leguminosarum with peas (Pisum), R.phaseoli with beans (Phaseolus), Bradyhizobium japonioum with soy (Glycine) and so on.

We have shown that bacterial genes (female ABC) that determine the early and early stages of genesis are controlled by ammonia: with increasing concentrations of ammonia · - the expression of nodABC genes is significantly reduced and

bowl formation (Dúsha et al., 1988: In Botba, H, de Bruijn, F.J., Newton, W (E <): Nitrogen Fixat: Hundred Years After, Gustav Fisher Verlag, Stuttgart, New York, p. 461),

We hypothesized that this regulatory process could be influenced by the expression of the riodABC genes at high ammonia concentrations.

According to the invention, the expression of nodABC genes at high ammonia concentration is improved by disabling a gene for Rhizobium melllotin that regulates the knockout gene expressor. This _guide is preferably made of the particular gene inactivity, such as by chemical mutagenesis or foreign DNA portion (e.g., transposon) the gene to be integrated * iodobenzonitrile deléoióval or any other suitable method.

In a preferred embodiment of the invention, the wild-type bacterial strain was mutagenized with the Tn5 transposon and searched for derivatives that expressed nodABC genes at a higher level than the wild-type bacterium as a result of the incorporation of the Tn5 transposon into the genome.

Selection was performed as follows:

In order to monitor the expression of the nodABC operon, the pRmM57 test plasmid was introduced by conjugation to IMulligan and Long (1985) Proc.Natl.Acad.Sci, USA 82, 6609-6613] in the Rhizobium meliloti strain [It.meliloti 1021]. This plasmid contains the nodABC operon, which contains a so-called "node" inserted into the nodC gene. reporter gene, the lacZ gene of Esch.erich.ia coli. The activity of the lacZ-encoded β-galactosidase enzyme can be easily measured (Miller (1972) Experiments in Moleoular Genetics, Cold Spring Harbor Foot, Press, New York), thus monitoring gene expression, another way to detect the bacterium to be tested is 5 bromo-4. grown on medium containing chloro-3-iudolyl-D-galactopyranoside (X-gal): if the cells have β-galactosidase activity, the colonies will turn blue.

Random Tn5 transposon mutagenesis using plasmid pSUP10111 as described by Simon et al. (19θ3) IMolecular Genetics of the

Bacteria-Piant Znteraction, Springer V _e rlag KG Berlin, pp 98-106]. Transposon-containing R.melilotl derivatives were selected on minimal medium (Kiss et al., 19791 (J.Gen.Miorobiol. 113, 105-118) containing kanamycin (200 µg / ml) and tetraolycin (10 µg / ml) for Tn5. and for the selection of the test plasmid. The resulting colonies were further examined on minimal medium containing 100 mM (NH 4) gSO 4, 10 µM luteolin, 0.04 mg / ml X-gal, and the above concentrations of kanamycin and tetraolycin. The resulting dark blue colonies were purified and further investigated The presence of luteolin, a host plant-derived fuvon, with the nodD gene is required for the induction of the nodABO operon {Mulligan and Long (1985) | (Proc.Nat.Aoad.S ₀ i. USA 82 , 6609-6613 Pétére et al. (1986: Scienoe, 233 * 977-980) · Introduction of the incompatible plasmid pPHUI from a strain having the appropriate mutant phenotype (L .: Mutant properties ¹ ') [Beringer (1978), Natúré 276, 633 -6341 el volltottuk test plazmldot (pRmM57) · The resulting mutant derivative strains of Rhizobium melilotl 399.

It should be noted that other mutagens that are incorporated into the gene may be used, such as another transposon.

The effective nodulation ability of strain R, melilotl 399 can be transferred to other Rhizobium / Bradyrhizobium species: If a specific Rhizobium / Bradyrhizobium species,

in which, like Rhizobium meliloti 1021, there is a nitrogen control of nodulation genes, and

1/1: a DNA sequence homologous to the 399 mutation region of R.meliloti can be detected, and the effective nodulation ability can be transmitted:

1.1.1 by mobilizing a given chromosomal region with conjugative plasmids (eg R68.45, RP4) in another Rhizobium / Bradyrhizobium species, where the mutant region can be incorporated into the homologous region of the reoipiens by homologous recombination,

1.1.2 transmission of the region to other R.meliloti strains transducing Rhizobium phages,

1.1.3 after molecular cloning of the mutation region, the DNA region can be inserted

a) conjugative plasmids (eg Ró8.45, RP4),

b) non-fconjugative but mobilizable plasmids (eg, pRK 290, pVK101, etc.),

c) introduction of the "suicide" plasmids and subsequent introduction of the recombinant plasmids into the reoipiens species by exchange of the mutant region and the wild-type homologue;

1/2: if homology is low but in vitro

It can also be detected by DNA-DNA hybridization: the partially homologous region can be isolated from a clone bank of the particular bacterial species, mutagenesis in Escherichia coli (which can be any insertion element with insertion or a

mutation clones) to return the mutant clones to the wild type strain.

Bacterial derivatives with similar properties to R.meliloti strain 399 can be prepared from other Rhizobium / Bi- adyrhizobium species by adapting the method described above:

if a homologous region is not detectable in a particular Rhizobium / Bradyrhizobium species but the nodulation genes are under nitrogen control, then a derivative can be selected by the method used in R.meliloti 1021 (random transposon mutagenesis in the presence of a nodal la fusion test plasmid), which expresses your female genes at a higher level in the presence of ammonia than the wild type strain.

Properties of mutant Rhizobium meliloti 399:

1. The mutant carries the transposon Tn5 in a single copy, integrated into the bacterial chromosome.

2. The growth rate of the mutant in the presence of various nitrogen sources (ammonium sulphate, potassium nitrate) is the same as that of wild type bacteria.

3. The expression of nodABC genes in the mutant is about half at low ammonia concentration and about half at high ammonia concentration. twice the wild-type bacterium • ·

riumban (Table I, Table A). Measured under the same conditions, it is not controlled by ammonia! gene expression does not change compared to wild-type control.

4. The expression of the nodD3 regulator gene in the mutant is similar to that of the wild-type bacterium, as is the expression of nodABC (Table B).

5. Control of the ammonia-regulated glutamine synthetase II gene was also altered in the mutant: the gene was not repressible with ammonia (Table I, C).

6. Known elements of the central nitrogen regulatory (ntr) system are not affected by Tn5 incorporation in the mutant: DNA-DNA hybridization results exclude mutation in the ntrC, ntrA or glnB genes. The mutant phenotype also excludes the ntrB gene (growth of R.meliloti 399 on nitrate is similar to that of wild type bacteria, whereas the ntrB: Tn5 derivative would not grow on nitrate) ·

7. The mutant bacterium has different abilities than the wild-type strain in the symbiosis with the host plant (Medicago sativa) when the medium contains bound nitrogen. In the presence of 2 mM, plants infected with the mutant bacterium produce more tubers than the wild type. Difference from day 9 after infection

by day 15 is statistically significant. Not only the number of tubers but also the number of nodulated plants were higher during the indicated period (Figure 1). To determine the total number of nodules and the number of nodulated plants, 30 to 30 sterile bred seedlings were infected with wild-type and nutant strains each, and 30 plants were grown uninfected as controls. After day 7, in a test tube on Gibson's medium [Gibson and Uutman (1960): Ann.Bot. The number of tubers on 24 »420-4331 growing alfalfa plants was determined daily. The same series was prepared on 2 mM Gibson medium containing NH 3 PgSO 4. The experiment was repeated twice.

8. Rhizobium bacteria, which form a bulb, can be isolated from the roots of alfalfa plants under sterile conditions. If a strain of bacteria tested has a marker, it can be determined, after proper selection, whether it has been involved in glioma formation.

By inoculating alfalfa plants grown in sterile conditions in a test tube with a mixture of wild-type and mutant bacteria, it is possible to determine the proportion of gap in nodulation based on the kanamycin resistance of R.meliloti strain 399 (a marker carried by Tn5).

10-10 plants were infected with R.meliloti 1021 and R.meliloti 399 and 1: 1 ratio of the two strains ···

(strains previously grown to the same optical density). After 1 month, bacteria were isolated from the tubers after purification by alcohol sterilization, and colonies were examined on selective (200 µg / ml kanamycin) and non-selective media. The colonies of plants infected with wild-type strains were grown on almost selective media. The colonies obtained from the tubers of the plants infected with the mutant strain were all kanamioin resistant. Plants infected with the mixture also produced tubers formed by the mutant bacterium; all 4θθ colonies were kanamioin resistant. The bound nitrogen-free medium containing 111. 2 mM (NH 2 SO 2) had the same result, the mutant strain inducing earlier tuber formation being more labile than wild-type bacteria under laboratory conditions.

9, The dry matter content (determined one month after infection) of the plants infected with the mutant bacterium was significantly higher than that of the plants infected with the wild-type strain (Table II). To determine the dry matter content, 500-500 plants (after germination in 5 18x18x18 cm pots containing 100 specimens each) were infected with wild-type or mutant strains and grown without infection. One series with Gibson nitrogen-free medium

and the other with Gibson's medium containing 2mM (NH 4) gSO 4 (50 ml / n / well). Plants were grown in a greenhouse (16 hours illumination, 20-22 ° C) · After one month, the shoots above the cotyledons were cut, 10-10 plants were grouped in a dish for 20 hours at 100 ° C and the weight of the plants was measured (II , spreadsheet).

The average dry weight of the plants grown in each pot _; (5 data for each sample) were analyzed by significance analysis. The dry weight of plants infected with wild-type bacteria was found to be significantly higher than that of the uninfected control (0mM (NH ^ j ^O SO: P = 5%, 2mM (NH ^ ^O SOSO ^: P = 10)). %), the dry weight of the plants infected with the mutant bacterium was significantly higher than that of the plants infected with the wild-type bacterium (P = 10 Jo in both cases).

TABLE I

Bacterial strain Test plasmid B-galactosidase activity (Miller unit)

3mM -L ^a + L ^a 100 mM -L ^{a m} v ₂ ^s0 4 + L ^a THE. R.meliloti 1021 θ, 7 0.4 - - R.meliloti 1021 _P RmM57 ^b 13 354 9 R.meliloti 399 pRmM57 ^b 19 182 11 140 R.meliloti 1021 pRmM6l ^c 714 577 550 388 R.meliloti 399 pRmM 61 ° 740 575 708 391 B R.meliloti 1021 pNTD I2 ^d 36 130 15 16 R.meliloti 399 pNTD 12 ^d 42 98 19 44 0.2% kno ₃ 0.2% (NH ₄ ) ₂ SO ₄ C. R.meliloti 1021 pFBó901 :: MudIIPR48 ^e 399 45 R.meliloti 399 pFB69O1 :: MudIIPR48® 259 198

^a * -L: Activity measured without luteolin + L: Activity measured after addition of luteolin * Plasmid pRmM57: fused to nodC-lacZ ^c 'pRmMó1: fused to nodD-lacZ ^d ' pNID12 plasmid: nodD3-lacZ fused to ® 'pFBud901s: -lacZ fusion

II. TABLE average dry weight / 10 plants

0 mM ₉ so ² 4 2 mM salt, 4 uninfected control 48.07 mg 100 / 0 - 159.02 Mg 100 <- / 0 - R.meliloti By 1021 fit. 80.12 mg 167 % 100 % 200.9 mg 126 % 100% R.meliloti 399-a

fit

108.49 mg

226% 135% 248.0 mg 156% 123%

Claims (4)

PATENT CLAIMS 1, A method for improving the expression of tubular binding genes in a high concentration of ammonia in a symbiotic nitrogen-binding bacterium, wherein the microorganism regulating its nodulatory gene expression is inactivated · 2. A method according to claim 1, characterized in that the gene is chemically mutagenized by inserting a foreign DNA sequence into the gene (insertion, transposition), deletion of the gene or a portion of the gene or deletion of a mutant gene. by transferring from strain to other strains or other species (conjugative or mobilizable plasmid amides, transducing phage, any plasmid or phage vector after molecular cloning) and inactivating them with the homologous region of the mutant gene sequence. 3. The method according to claim 1 or 2, wherein the wild-type bacterial strain is mutagenized with Tn5 transposin tin. The method according to claim 3, wherein the bacterial strain which expresses nodulatory genes at a higher level in the presence of ammonia is selected after nrutagenesis.